1. Field of the Invention
The present invention relates to a protein chip substrate and a method for manufacturing a protein chip substrate. More specifically, the present invention relates to a method for manufacturing a protein chip substrate wherein plasma polymerized ethylenediamine (PPEDA) having an amine group is deposited on plasma polymerized cyclohexnane (PPCHex) being free of non-specific adsorption by inductively coupled plasma-chemical vapor deposition (ICP-CVD), thereby preventing non-specific adsorption of proteins on a slide surface, and a protein chip substrate manufactured by the method.
2. Description of the Related Art
With recent discoveries of human genomic structures, research for human genomic functions has increasingly attracted considerable attention.
There have been advances in a new research field referred to as “functional proteomics” for systematically probing functions of human genes.
Functional proteomics is divided into three groups of “genomics”, “proteomics” and “bioinformatics”. Genomics is focused on studies targeting genes, proteomics is focused on studying the behavior of genes by targeting overall proteins in cells, and bioinformatics is a combined-approach of the two fields.
Molecular biological access to function studies for genes or cells was made on the basis of controlling a single gene or mRNA expressed by the gene. Nowadays, the recent trend towards proteome-based analysis is being introduced.
Protein chips are required for the proteome-based analysis as a key technique (Gavin MacBeath and Stuart L. Schreiber, Science, 289:1760˜1763, 2000).
The protein chips are an automatic system capable of simultaneously analyzing binding structure of a large amount of proteins by immobilization of tens to thousands of proteins on a small substrate.
The protein chips are similar to microarray DNA chips which detect gene expressions or mutants by immobilizing tens to thousands of genes on a small substrate, and simultaneously analyzing variations in a large amount of genes through the behavior of complementary bindings between genes (Yasuda, H., Lamage, C. E., J. A ppl. Polym. Sci. 17: 201, 1973; Muguruma, H., Karube, I., Trends Anal. Chem. 18: 62, 1999; Nalanishi, K., Muguruma, H., Karube, I., Anal. Chem. 68: 1695, 1996; Miyachi, H., Hiratsuka, A., Ikebukuro, K., Yano, K., Muguruma, H., Karube, I., Biotechnol. Bioengin. 69: 323, 2000).
However, protein chips can be used for the following analysis unattainable by DNA chips.
First, they can be used to obtain information regarding interaction between protein-protein. The signal transmission or control within cells is represented as the interaction between protein-protein. Thus, this can be analyzed by protein chips.
Second, proteins, from higher organisms including humans to yeasts, undergo post-translational modification. As a result, it is possible to obtain information regarding a secondary modification such as phosphorylation and oxidation.
Third, proteins enable detection of problems which may be generated after mRNA formation. A variety of diseases are caused by problems associated with post-transcription control, protein generation and protein localization. DNA chips used to detect mRNAs make it difficult to obtain information regarding the problems.
Fourth, when proteins are expressed from mRNAs, correlation in quantity between the proteins and mRNAs is often not sufficiently high. Thus, in some cases, DNA chips have limitation of difficulty in obtaining correct information associated with proteins. However, protein chips enable solving such limitation of DNA chips.
Fifth, difference in base sequence of genes revealed by DNA chips is not directly indicative of disease or difference in phenotype. Furthermore, since mutants derived from amino acids having similar characteristics generally retain inherent characteristics of proteins, they hardly cause disease or difference in phenotype.
On the other hand, the use of protein chips enables identification of the protein inducing disease or difference in phenotype.
In addition, the use of protein chips ensures database formation for relationship between protein-protein, and is effective in reducing time and costs upon development of novel medicines. For example, a period required to develop a treatment medicine using protein chips can be considerably shortened, as compared to the cases using current genes.
Protein chips are utilized in a wide variety of industrial applications including disease diagnosis, environment monitoring and harmfulness testing, as well as industrial applications.
Based on the mentioned advantages, protein chips are expected to have high marketability, as compared to DNA chips. Thus, there is an eager demand for developing protein chips.
Such a protein chip includes a sensor chip and a protein detection system.
The sensor chip is a major constituent for determining a developing rate of the protein chip.
The sensor chip has a structure in which tens to thousands of proteins are arranged in a predetermined array on the surface of a small chip. The chip is selected from a slide-sized glass or plastic.
A key technique associated with the sensor chip is protein immobilization for attaching proteins to the surface thereof.
The protein immobilization techniques are classified into three groups, based on the characteristics.
First, immobilization of a specific protein to the surface of sensor chips using carboxymethyl-dextran (CM) is the most widely used method. Here, amine coupling is the most commonly used. Alternately, thiol coupling or avidin-biotin coupling is used to immobilize acid proteins or DNAs.
Second, surface treatment of sensor chips is suitable for immobilizing a plurality of protein groups having the same characteristics.
Third, poly lysine or calyx crown is available for immobilizing large amount of non-specific proteins.
The control of protein immobilization and patterned array of biomolecules are implicated in wide a variety of fields including basic studies of biochips, bioelectronics and cell-biology.
A variety of techniques, e.g., self-assembled monolayer and lithography, are generally used for protein immobilization.
In recent years, studies for immobilizing bio molecules on a solid slide using plasma polymerized films have been actively investigated.
Plasma is a state in which neutral gas molecules absorb electrical or thermal energy, and are then separated into ions and electrons.
Studies associated with techniques utilizing plasma have been actively made.
Plasma is being gradually expanded to a variety of applications including plasma etching and plasma enhanced chemical vapor deposition (PECVD) in semiconductor device fabrication, surface treatment of metals or polymers, synthesis of enhanced materials (e.g., imitation diamonds), plasma display panels (PDPs) and environment protection techniques.
Plasma partially ionizes gas, and efficiently activates molecules.
Relatively inert molecules are exposed to plasma, thereby being readily activated.
Coating of plasma polymerized films is achieved by arranging monomers in a deposition chamber, activating or decomposing reactants with plasma, and allowing the reactants to be condensed on the surface of a slide, to form a polymerized film.
When precursors containing an amine or aldehyde group are used, plasma polymerized films can contain a large amount of the group. Thus, such solid slide is utilized in biomolecules immobilization.
The plasma polymerized films are largely different with chemically-polymerized general films.
Such plasma polymerized film has no porosity, is mechanically and chemically stable, and has an adhesive property to the slide due to its cross-linked structure.
In addition, the plasma polymerized film has superior controllability in thickness and high uniformity, as compared to chemically-polymerized films. These advantages provide suitability for manufacture of protein and DNA arrays.
To achieve highly sensitive diagnostic protein chips, a sufficient amount of proteins must be immobilized at a predetermined space thereof. Accordingly, there is a demand for methods that can immobilize proteins more efficiently.
Protein immobilization based on covalent bonds depends on nucleophilicity or electrophilicity due to chemical or physical formation on the surface of the slide glass.
A well-known problem in experiments associated with protein chips is non-specific adsorption of proteins on the surface of the slide glass. The non-specific adsorption is misrecognized as obviously false response in diagnostic inspection.
However, conventional protein chips have a problem of occurrence of non-specific adsorption even in a region where no amine group exists.
Thus, there is a need for techniques that minimize non-specific adsorption upon diagnosis based on protein chips.